a) Field of the Invention
This invention is related to methods for rapid hybridization of complementary nucleic acid molecules and their use in purification, immunoassays, biosensors, and other biochemical applications.
b) Description of Related Art
The process by which one member of a pair of nucleic acids (oligonucleotides, DNA, RNA) binds its complementary member is known as hybridization. Since nucleic acid molecules have a very strong preference for their sequence complements, simple mixing of complementary sequences is enough to induce them to hybridize. Hybridization is temperature dependent, so nucleic acid molecules that hybridize strongly at low temperatures can be temporarily separated (denatured) by heating. Hybridization is the basis for the polymerase chain reaction and in situ hybridization technologies.
Hybridization is a two step process involving (1) diffusion of the target nucleic acid molecule through the hybridization medium until it reaches an immobilized probe and (2) contact with the probe and binding to its complementary sequence. This process is usually slow because the limited amount of target takes very long to diffuse to the probe surface. The diffusion rate is usually increased by increasing the temperature. Although this increases the hybridization rate, there are limits above which the molecules may be irreparably harmed and the process still requires inordinate amounts of time to complete. Numerous parties have addressed the problem of decreasing cycle time in thermal cyclers and related devices.
The actual hybridization reaction represents the most important and central step in the whole process. The hybridization step involves placing the prepared nucleic acid sample in contact with a specific reporter probe, at a set of optimal conditions for hybridization to occur to the target sequence. Hybridization may be performed in any one of a number of formats. For example, multi-sample nucleic acid hybridization analysis has been conducted on a variety of filter and solid support formats (See G. A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, N.Y., Chapter 19, pp. 266-308, 1985). One format, the so-called “dot blot” hybridization, involves the non-covalent attachment of target nucleic acids to filters, which are subsequently hybridized with a radioisotope labeled probe(s). “Dot blot” hybridization gained wide-spread use, and many versions were developed (see M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridization—A Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp. 73-111, 1985). It has been developed for multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for the detection of overlapping clones and the construction of genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).
New techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions of the conventional “dot blot” and “sandwich” hybridization systems. The micro-formatted hybridization can be used to carry out “sequencing by hybridization” (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992).
U.S. Pat. No. 5,849,486 teaches the use of electric fields to accelerate hybridization by bringing the negatively charged target molecules close to the immobilized probes. This greatly accelerates hybridization but requires a restricted flow area due to the tendency of the negatively charged DNA to follow the electric field lines, which do not necessarily cross the complementary target DNA location. In order to overcome this problem, the probe DNA has been immobilized within the pores of a polymer such as agarose. Even though this brings the target DNA and the probe DNA in close contact, the movement across the restricted pores slows down the probe because of the high hydrodynamic resistance.
This approach is also nonspecific in nature since the movement of the probe DNA in the electric field depends on the molecule's charge, which varies from molecule to molecule depending upon its sequence. Another problem with this approach is that the metal used in the manufacturing of the electrodes, which generate the electric field, may become magnetized.
The object of the present invention is to provide an improved method for the rapid hybridization of nucleic acids on solid surfaces. Several longstanding problems in purification, immunoassays, biosensors, and other biochemical applications are resolved by this invention.